Chitosan Oligosaccharides Stimulate the Efficacy of Somatic

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Article Chitosan Oligosaccharides Stimulate the Efﬁcacy of Somatic Embryogenesis in Different Genotypes of the Liriodendron Hybrid

Asif Ali 1, Jiaji Zhang 1, Minmin Zhou 1, Tingting Chen 1, Liaqat Shah 2, Shams ur Rehman 1, Sikandar Hayat 3, Jisen Shi 1 and Jinhui Chen 1,*

1 Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education of China, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; [email protected] (A.A.); [email protected] (T.C.); [email protected] (S.u.R.); [email protected] (M.Z.); [email protected] (J.Z.); [email protected] (J.S.) 2 Department of Botany, Mir Chakar Khan Rind University, Sibi Baluchistan 82000, Pakistan; [email protected] 3 Department of Landscape Plants, College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, China; [email protected] * Correspondence: [email protected]

Abstract: Liriodendron hybrid (L. chinense × L. tulipifera), an essential medium-sized tree generally fa- mous for its timber, is also used as an ornamental and greenery tool in many places around the world. The Liriodendron hybrid (L. hybrid) tree goes through many hurdles to achieve its maximum strength and vigor, such as loss of habitat, vast genetic variation, and low seed setting rate. The establishment of an effective and well-organized somatic embryogenesis (S.E.) system could be used to overcome Citation: Ali, A.; Zhang, J.; Zhou, M.; these obstacles, rather than the old-fashioned seed culture and organogenesis. This study is based Chen, T.; Shah, L.; Rehman, S.u.; on the impact of chitosan oligosaccharide (COS) and its role in the induction of S.E. on the callus of Hayat, S.; Shi, J.; Chen, J. Chitosan L. hybrid Oligosaccharides Stimulate the four genotypes of the . The optimal concentration of COS could enhance the momentum Efﬁcacy of Somatic Embryogenesis in and effectiveness in S.E.’s mechanism, which further improves the growth rate of the L. hybrid tree’s Different Genotypes of the plantlets. This study shows that COS has a prominent role in endogenous hormones like indole acetic Liriodendron Hybrid. Forests 2021, 12, acid (IAA), zeatin (Z.T.), and gibberellic acid (GA3). Furthermore, COS improves the growth devel- 557. https://doi.org/10.3390/ opment, growth speed, as well as the development situation of plant germination ability. COS can f12050557 also regulate branch development and root growth, which could be linked to the antagonistic effect on growth factors to some extent or by affecting auxin synthesis and polar transport. Academic Editor: Donald L. Rockwood Keywords: Liriodendron hybrid tree; somatic embryogenesis; chitosan oligosaccharides; IAA; ZT; GA3

Received: 19 March 2021 Accepted: 21 April 2021 Published: 29 April 2021 1. Introduction L. hybrid Liriodendron Publisher’s Note: MDPI stays neutral The tree, also known as the hybrid, is a large growing tree species with regard to jurisdictional claims in in the East Asia region. L. hybrid, produced from the cross between Liriodendron chinense published maps and institutional affil- (Hemsley) Sargent and Liriodendron tulipifera Linn, is considered a vicarious species [1]. iations. However, little has been known about its genetic divergence and evolutionary trajecto- ries [2]. The hybrid is primarily grown for its timber quality and landscaping purposes [3] and is one of the most abundant tree species in China’s temperate regions. The hybrid is a separate species and has distinct morphological and biological characteristics that have been widely cultivated and used in China [4]. Typically, the genus of Liriodendron belongs Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. to Magnoliaceae, which occupy a critical evolutionary position [5–11]. These two relict This article is an open access article species of Liriodendron have been suggested to be divaricated in the early ages during the distributed under the terms and middle to late Miocene [2,12]. It is an excellent garden ornamental and garden cultivation conditions of the Creative Commons tree species and grows fast with light and soft wood and is cultivated in many temperate Attribution (CC BY) license (https:// mountains of America and China for wood production [13–17]. The tree is tall, the trunk creativecommons.org/licenses/by/ is perfect and straight, the wood structure is fine and uniform, the color is light, easy to 4.0/). dry, and easy to process, and the fiber is longer. In the sexual reproduction of L. chinense,

Forests 2021, 12, 557. https://doi.org/10.3390/f12050557 https://www.mdpi.com/journal/forests Forests 2021, 12, 557 2 of 17

seed setting percentage is very low, and it is considered its prominent trait. In a natural environment, the seed setting ratio of Liriodendron is very low and is not more than 10% [9]. It is an excellent tree species for pulp, paper, artificial board, and furniture. It is suitable for afforestation in mountains and hills and is also a remarkable tree species for building industrial raw material forests. Furthermore, Liriodendron is valued as a source material for honey production, chemical extract [18–20], and biofuels [21,22]. Liriodendron chinense (Hemsl.) Sarg. (L. chinense) has been classified as a very rare and endangered species because of its limited occurrence [23]; therefore, back in 1992, L. chinense was listed in the Red List of Endangered Plants in China [24]. Later on, in 1998, the International Union for Conservation of Nature and Natural Resources has enlisted L. chinense as a near-threatened species in the IUCN Red List of Threatened Species. S.E. is a synthetic developmental technique of acquiring a plant or embryo from a single somatic cell. Under appropriate conditions, this newly formed embryo can further develop into a whole plant [25]. S.E. is a stable and more reliable tool, and this process, controlled by the relevant genes, has a vital role in the entire development of somatic embryos [26]. The primary step in acquiring the embryo cell involves the transition of cell fate from a somatic cell. S.E. has widely been practiced in many woody plants and others by directly introducing somatic cells, such as in Bacopa monnieri (L.) Wettst [27], or indirect induction through the callus stage, as in Gentiana decumbens L.f., Theobroma cacao L. [28], and Ledebouria revolute [29]. S.E. has a vital role in clonal propagation in woody plants and is an essential source for synthetic seed production, germplasm conservation, and cryopreser- vation [25]. Moreover, S.E. has a significant role in mass propagation in vitro, germplasm conservation, and woody plants’ genetic improvement [30–32]. In vitro propagation not only plays a significant role in the clonal propagation of desirable genotypes, but it could also provide suitable target material for genetic transformation [33,34]. Many artificial growth regulators negatively affect the eco-friendly environment; therefore, natural polysaccharides could stimulate plant growth and development [35,36]. Chitin, a fibrous substance comprising polysaccharides, is an abundant biopolymer [37]. Chitin is one of the main components of invertebrate exoskeletons and fungi cell walls [38]. In the past, chitin and its deacetylated product chitosan have been widely used to improve agricultural crop plants in various ways [39–41]. The structure of chitosan contains 2-acetamido-2-deoxy-β-d-glucose (NAG) monomers linked together by β (1→4) link- ages [37]. When the degree of acetylation (DA) (expressed as a molar percentage), used to differentiate chitin from chitosan, is lower than 50 mol%, the product is named chitosan [42]. Oligosaccharides have been reported earlier, in 1983 [43], through enzymatic hydrolysis of the plant cell wall and different crop plants. Besides, it has positive results when applied exogenously [44,45]. In plants, oligosaccharides can be used as a growth regulator for development and survival in the ecosystem [46]. Chitosan oligosaccharides’ (COS) hydrolysis of chitosan or chitin has been paid extensive attention in recent years. This is mainly due to their physicochemical properties like lower molecular weight, lower viscosity, higher water solubility, biocompatibility, and biodegradability [47,48]. Coating the bulbs of Ornithogalum saundersiae with COS enhances the growth vigor, early flowering, and improves the content of pigments, polyphenols, L-ascorbic acid, potas- sium, phosphorus, zinc, and manganese [49]. COS, carrageenan, alginate, and their de- polymerized derivatives could positively enhance plant growth and development [49–51]. Exogenous treatment of oligosaccharides can stimulate fruit ripening in Lycopersicon escu- lentum (Mill.) and improve natural fruit softening [52]. Spraying COS at the tillering stage increases the yield in wheat [53]. COS induces fruits’ antioxidant activity and improves the fruit texture, quality, and taste in strawberries [54]. Due to its non-toxic and high solubility properties, COS has been the subject of increased attention in terms of its pharmaceutical and medicinal applications [55]. Although much research has shown the significance of COS involved in plant growth and development, the concise mechanism of COS on plant S.E. has not been studied in detail. Forests 2021, 12, 557 3 of 17

Our primary studies showed the improvement in the efficiency of S.E. of L. hybrid. More specifically, we have demonstrated the analyzed data indicating the significant amount of development in the germinated seedlings grown from somatic embryos. Fur- thermore, we have shown the effect of COS and obtained promising results that show the increased efficiency of S.E. of L. hybrid. Lastly, we examined endogenous hormones’ influence during the formation of somatic embryos treated with COS.

2. Materials and Methods 2.1. Plant Material and Culture Initiation In this study, we used the embryogenic callus of L. hybrid, and the reagent used was of COS in the basic 3/4 MS solid medium. The genotypes used were callus 154102, 155202, 166302, and BxB. The four callus lines all belong to the same species, i.e., Liriodendron hybrid, but they differ based on time, and their parental lines are different. Genotypes 154102 and 155202 originated in 2015, while genotypes 166203 and BxB were created in 2016 and 2019, respectively. All the calli were yellowish, dense, and ﬁne, with a moderate growth rate and stable state. Callus of L. hybrid is a good combination of L. hybrid bred by the Nanjing Forestry Univer- sity research group from 2012 to 2014, induced by the immature embryo. The medium used before treatment was M13, 3/4 MS + 2,4-d 1.0 mg L−1 + 6-ba 0.2 mg L−1 + VC 5.0 mg L−1 + Sucrose 30 g L−1 + CH 0.5 g L−1, pH: 5.7–5.8. MS denotes Murashige & Skoog, BA is 6-benzylaminopurine, VC indicates vitamin C, and CH is casein hydrolysate.

2.2. Conﬁguration of COS Stock Solution COS is easily soluble in water, and its autoclaved activity remains unchanged. The product of chitosan oligosaccharide was bought from Sinopharm Chemical Reagent Co., Ltd. (Catalogue No. TCI-C2849). The solution of chitosan oligosaccharide was prepared in the Nanjing Forestry University laboratory by diluting 1 g of the product in 20 mL of double-distilled water in a beaker. The volume is ﬁxed to 1 L, which is the stock solution of COS with a concentration of 1 mg mL−1. Finally, it is transferred to a brown bottle and stored and kept away from light at 4 ◦C for later use. Solution and culture media were autoclaved at 120 ◦C for 20 min, and then transferred to a brown bottle and stored and kept away from light at 4 ◦C for later use.

2.3. Concentration of COS The somatic embryo induction medium of L. hybrid was prepared using 3/4 MS as the basic medium and adding different COS concentrations. COS-free 3/4 MS basic medium with ABA (abscisic acid) was used as a control. Four genotypes with three concentration gradients set were used in the experiment. Each concentration was inoculated with at least three dishes, and each dish was connected with ﬁve calli, where each callus was about 0.5 cm in diameter and 1.6 g/dish in weight (Table1). The initial culturing environment was 24 ◦C dark culture.

Table 1. Culture medium with different concentrations of COS.

Culture Medium No. Components of Culture Medium 1 3/4 MS + VC5 mg L−1 + Sucrose30 g L−1 + ABA 2 mg L−1 2 3/4 MS + VC5 mg L−1 + Sucrose30 g L−1 + COS 0.01 mg L−1 3 3/4 MS + VC5 mg L−1 + Sucrose30 g L−1 + COS 0.05 mg L−1 4 3/4 MS + VC5 mg L−1 + Sucrose30 g L−1 + COS 0. 1 mg L−1

2.4. In Vitro Growth of Somatic Embryo Seedlings The somatic embryo seedlings were transferred into the 3/4 MS medium without plant growth regulators to enhance the growth of regenerated seedlings. The regenerated plantlets were cultured under a photoperiod of the 16 h light/8 h dark at 25 ◦C. Forests 2021, 12, 557 4 of 17

2.5. Ex Vitro Adaptation of Somatic Embryo Seedlings All the regenerated plantlets were transferred to the pots (plastic pots) after eight weeks on a growth medium filled with vermiculite:perlite:soil (1:1:3, v/v). The plantlets were carried to the greenhouse to acclimatize under a 16 h photoperiod at 120 µmol m−2 133 s−1 provided by cool white ﬂuorescent tubes at 21 ± 2 ◦C and relatively high humidity (70–90%).

2.6. Somatic Embryo Observation For induced somatic embryos of L. hybrid, stereoscopic and optical microscopy were used to observe the somatic embryos. Photographs were taken to study the morphological formation and induction during the S.E. of each genotype different media. To avoid errors caused by callus block size, the somatic embryo induction amount was determined using the mass method. The number of somatic embryos induced by callus per unit mass was taken as the statistical object to analyze the optimal concentration of COS for inducing somatic embryos on a solid medium.

2.7. Acquisition and Growth Potential Measurement of Regenerated Plants After the embryoid body grew from the callus’ surface and developed into the cotyledon embryo stage, the culture conditions were adjusted. The material was transferred to the tissue culture bottles in the light culture room, and the regenerated plants were further cultured in the 3/4 MS basic medium. One month later, using the 3/4 MS basic medium as a control, the differences in regeneration rate, branching rate, and plant height of the regenerated plants were compared. For the plants that produced branches, they were divided into three modes according to the number of branches, the total number of leaves (TNL), total stem length (TSL), the average number of leaves per branch (LN/B), and average stem diameter per branch (BD/B) of branches 1, 2, and 3, respectively. When the regenerated seedlings grew up, they were domesticated and transplanted into small pots with a 5–8 cm diameter. To understand the plant growth more clearly, the statistical standard was formulated as the following formula:

Seedling rate = (No. of regenerated somatic embryos)/(No. of somatic embryos) × 100%

Branching rate = (No. of branches)/(No. of total plants) × 100%

2.8. Seedling Effects of COS To verify whether COS has a strong effect on seedling of L. hybrid, about 60 seedlings with the same basal stem and growth from the same genotype were selected, and 30 days of the seedlings were divided into four groups and cultured in 3/4 MS basic medium supplemented with 0.01, 0.05, and 0.1 mg L−1 COS. After two months, plant growth of the two groups was compared, and plant height, stem length, stem diameter, leaf size, root length, and root diameter were statistically determined.

2.9. Analysis of Endogenous Hormone Levels The embryogenic callus of COS- and ABA-induced somatic embryos for all the four genotypes was randomly quantiﬁed to check endogenous hormones such as control (C.K.), ABA (2 mg L−1), and COS. The samples were randomly taken at days 0, 15, 30, and 60 after different treatments to detect endogenous hormones. Besides, the samples were immedi- ately frozen in liquid nitrogen and stored at −80 ◦C until analysis. The samples treated without any plant growth regulators were selected as the controls.

3. Data Analysis SPSS 16.0 statistical software was used for one-way ANOVA, and Duncan’s multiple tests were used to compare the mean values. The signiﬁcance level was set as alpha = 0.05 indicated as * and alpha = 0.01 denoted as **. GraphPad Prism was used to plot the analysis results of SPSS software. Forests 2021, 12, 557 5 of 17

4. Results COS improves the efficacy of S.E. The embryogenic callus of four genotypes of L. hybrid was cultured on the medium containing 0.01, 0.05, and 0.1 mg L−1 COS to study its impact and observe whether COS regulates somatic embryos’ induction. The number of somatic embryos induced in the callus of all four genotypes was significantly different in different concentration media (Figure1). In the embryogenic callus of all four genotypes, the total number of somatic embryos produced by the medium having 0.05 mg L−1 COS was more than twice as compared to the control group (Figure1). The number of mature somatic embryos induced by COS (0.05 mg L−1) for all the genotypes was significantly higher than the control (Figure2 ). The number of total somatic embryos and mature somatic embryos induced by the medium with 0.05 mg L−1 COS was significantly higher than that induced by the two other media containing 0.01 and 0.1 mg L−1, as well as with the control group. A low concentration of 0.05 mg L−1 of COS could promote S.E. of the callus of L. hybrid, while a high concentration of COS could inhibit the induction of S.E. In the genotype 166302, when the COS concentration was 0.05 mg L−1, the average number of somatic embryos produced per gram of callus was the highest, up to 2456 on average, more than two times that of the control group (Figure1d). When the concentration of COS was >0.05 mg L−1, the somatic embryo induction ability was significantly decreased. Besides, mature somatic embryos’ occurrence was consistent with the trend of overall embryos, and the number reached the highest at 0.05 mg L−1 for all the genotypes. Therefore, the optimal concentration of COS for S.E. of all the four genotypes is 0.05 mg L−1. Compared with the control, the embryogenic callus of the L. hybrid treated with COS is relatively good, with a pale-yellow color, moderate water content, and a more significant number of S.E.; however, the callus of the control group showed uneven status (Figure1a–d). Some calli grew well and could produce somatic embryos, while the other calli showed browning and water-stained shape and could not produce somatic embryos or rarely produced deformed embryos. The number of mature somatic embryos of genotype 166302 is significantly higher than that of other genotypes, which could have three reasons (Figure1g). First, genotype 166302 itself has a more robust S.E. and development ability than the other genotypes. Secondly, it is more sensitive to COS than the other genotypes; therefore, the somatic embryo induction effect of COS can be fully utilized. Third, the callus particles were smaller than the other genotypes; thus, more callus particles per unit mass and the number of somatic embryos and mature somatic embryos are more extensive (Figure1a–g). The number of mature somatic embryos of genotype 166302 was higher at 0.05 mg L−1, and the number of mature somatic embryos is higher than that of genotypes 154102, 155202, and BxB. Its peak appears at a concentration of 0.05 mg L−1 (Figure2c). This study found that the number of mature somatic embryos was also the highest when the COS concentration was 0.01 or 0.05 mg L−1. The difference between the two concentrations was highly significant (Figure2). The total number of embryos induced by 0.01 or 0.05 mg L−1 was relatively high because this concentration was more suitable for the growth and development of somatic embryos of L. hybrid. There was no significant difference in the number of mature somatic embryos between the control and 0.1 mg L−1. Still, the number of somatic embryos was significantly different, indicating that high concentrations of COS may inhibit the growth and development of L. hybrid (Figure2). Unlike the total somatic embryos’ statistics, the number of mature somatic embryos of genotype 154102 was higher at 0.01 mg L−1. The number of mature somatic embryos of all four genotypes varies largely with different COS concentrations. The number of mature somatic embryos was higher when the concentration reached 0.01 and 0.05 mg L−1 for all the four genotypes, and its peak appears at a concentration of 0.05 mg L−1 (Figure2). Forests 2021,, 12,, 557x FOR PEER REVIEW 6 6of of 19 17

Figure 1. Several somatic embryos with different concentrations of COS. The ( a–d)) represent represent ( (e–h)) at at the the interval interval of of 0, 0, 15, 15, 30, Forests and 60 2021 days, 12, respectively,x FOR PEER REVIEW ofof genotypegenotype 166302.166302. TheThe signiﬁcancesignificance levellevel waswas setset asas alphaalpha == 0.050.05 indicated as ** andand alphaalpha7 of ==19 0.01 denoted as **. GraphPad GraphPad Pris Prismm was was used used to to plot plot the the analysis analysis results results of of SPSS SPSS software. software. The The scale scale bar bar level level was was set set as as 2 2mm, mm, and and all all the the genotypes genotypes show show almost almost similar similar result results;s; therefore, therefore, we we represent represent only only genotype genotype 166302 166302 here. here.

The number of mature somatic embryos of genotype 166302 was higher at 0.05 mg L−1, and the number of mature somatic embryos is higher than that of genotypes 154102, 155202, and BxB. Its peak appears at a concentration of 0.05 mg L−1 (Figure 2c). This study found that the number of mature somatic embryos was also the highest when the COS concentration was 0.01 or 0.05 mg L−1. The difference between the two concentrations was highly significant (Figure 2). The total number of embryos induced by 0.01 or 0.05 mg L−1 was relatively high because this concentration was more suitable for the growth and development of somatic embryos of L. hybrid. There was no significant difference in the number of mature somatic embryos between the control and 0.1 mg L−1. Still, the number of somatic embryos was significantly different, indicating that high concentrations of COS may inhibit the growth and development of L. hybrid (Figure 2). Unlike the total somatic embryos’ statistics, the number of mature somatic embryos of genotype 154102 was higher at 0.01 mg L−1. The number of mature somatic embryos of all four genotypes varies largely with different COS concentrations. The number of mature somatic embryos was higher when the concentration reached 0.01 and 0.05 mg L−1 for all the four genotypes, and its peak appears at a concentration of 0.05 mg L−1 (Figure 2).

Figure 2.FigureThe total2. The embryos total embryos and matureand mature embryos embryos at differentat different COS COS concentrations concentrations for for all all the the four four genotypes genotypes of L. of hybrid,L. hybrid , (a–d) represent(a–d) represent 154102, 154102, 155202, 155202, 166302, 166302, and and BxB, BxB, respectively. respectively. The The signiﬁcance significance levellevel was was set set as as alpha alpha = 0.05 = 0.05 indicated indicated as * as * and alphaand = alpha 0.01 denoted = 0.01 denoted as **. as GraphPad **. GraphPad Prism Prism was was used used to to plot plot the the analysis analysis resultsresults of of SPSS SPSS software. software. All All four four genotypes genotypes show approximatelyshow approximately parallel parallel results. results.

4.1. COS4.1. COS Regulating Regulating the the Level Level of of Endogenous Endogenous HormonesHormones When the embryogenic callus of the genotype 154102 was cultured on a medium con- When the embryogenic−1 callus of the genotype 154102 was cultured on a medium taining 0.05 mg L −COS,1 the IAA content was significantly increased at days 15, 30, and containing60 (Figure 0.05 3a). mg After L 30COS, days theof culture, IAA content a large wasnumber signiﬁcantly of spherical increased embryos began at days to 15, 30, andappear, 60 (Figureand torpedo3a). embryos After 30 developed days of culture,around 30 a days, large and number a large of number spherical of globular embryos beganembryos to appear, were andcontinuously torpedo produced embryos (Figure developed 3b). After around 45 days 30 days, of culture, and a most large of number the of globularsomatic embryosembryos developed were continuously from the callus’s produced surface (Figure matured3b). early After and 45 could days be of trans- culture, ferred to the light culture room for cultivation to regenerate plants. On the other hand, many embryoid bodies were brewed inside and on the original callus’ surface, which continuously emerges and grows rapidly. Most cotyledon embryos were transferred to the light culture when they were mature at 60 days (Figure 4a–d).

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most of the somatic embryos developed from the callus’s surface matured early and could be transferred to the light culture room for cultivation to regenerate plants. On the other hand, many embryoid bodies were brewed inside and on the original callus’ surface, which Forests 2021, 12, x FOR PEER REVIEW 8 of 19 continuously emerges and grows rapidly. Most cotyledon embryos were transferred to the light culture when they were mature at 60 days (Figure4a–d).

Figure 3. IAA content of all four (a–d) genotypes induced by COS at the concentration of 0.05 mg L−1, and ABA at the Figure 3. IAA content of all four (a–d) genotypes induced by COS at the concentration of 0.05 mg L−1, and ABA at the concentration of 2 mg L−1, recorded after 0, 15, 30, and 60 days, respectively. CK represents control, while the signiﬁcance concentration of 2 mg L−1, recorded after 0, 15, 30, and 60 days, respectively. CK represents control, while the significance level waslevel set aswas alpha set as = alpha 0.05 indicated= 0.05 indicated as * andas * alphaand alpha = 0.01 = 0.01 denoted denoted as as **. **. GraphPad GraphPad Prism was was used used to toplot plot the the analysis analysis results ofresults SPSS of software. SPSS software.

Furthermore, for the genotype of 154102, the content of endogenous Z.T. significantly increased in the COS-treatment group before the stage of the globular embryo and then increased slightly in the subsequent development. Endogenous ZT’s content in ABA treatment was almost the same and was not different from that in COS, which had a slight drop trend at the early developmental stage (Figure5a). The contents of GA3 in the ABA treatment were initially significantly increased, while they decreased for the COS treatment after 15 days. After 30 days, an abrupt decrease was observed for GA3 for both ABA and COS treatments and the control group. At 60 days, it was increased significantly for both ABA and COS, with COS slightly higher than the rest of the treatments (Figure6a).

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Figure 4. Column-wise representation of somatic embryogenesis of all the four genotypes of L. Figurehybrid in 4. responseColumn-wise to COS 0.05 representation mg L−1 after 0, 30, of 45, somatic and 60 embryogenesisdays. Genotypes 154102, of all 155202, the four genotypes of L. hybrid − in166302, response and BxB to at COSdifferent 0.05 time mg intervals L 1 after of 0 (a 0,), 30 30, (b), 45, 45 ( andc), and 60 60 days. days (d Genotypes) were observed 154102, 155202, 166302, −1 andafter BxBtreating at differentwith COS 0.05 time mg intervals L . The scale of 0bar (a level), 30 was (b), set 45 as (c 2), mm, and and 60 all days the pictures (d) were were observed after treating captured on the same given day and time. −1 Forests 2021, 12, x FOR PEERwith REVIEW COS 0.05 mg L . The scale bar level was set as 2 mm, and all the pictures were10 capturedof 19 on the

sameFurthermore, given day and for the time. genotype of 154102, the content of endogenous Z.T. significantly increased in the COS-treatment group before the stage of the globular embryo and then increased slightly in the subsequent development. Endogenous ZT’s content in ABA treatment was almost the same and was not different from that in COS, which had a slight drop trend at the early developmental stage (Figure 5a). The contents of GA3 in the ABA treatment were initially significantly increased, while they decreased for the COS treatment after 15 days. After 30 days, an abrupt decrease was observed for GA3 for both ABA and COS treatments and the control group. At 60 days, it was increased significantly for both ABA and COS, with COS slightly higher than the rest of the treatments (Figure 6a).

−1 Figure 5. The ZTFigure content 5. The ofZT all content the four of all ( athe–d four) genotypes (a–d) genotypes induced induced by COSby COS at at the the concentration concentration of of 0.05 0.05 mg mg L−1 and L ABAand at ABA at the − concentration ofthe 2concentration mg L 1, recorded of 2 mg L− after1, recorded 0, 15, after 30, 0, and 15, 30, 60 and days, 60 days, respectively. respectively. CK CK represents represents control, control, while while the signifi- the signiﬁcance level was set ascance alpha level = was 0.05 set indicated as alpha = as0.05 * andindicated alpha as * = and 0.01 alpha denoted = 0.01 denoted as **. GraphPad as **. GraphPad Prism Prism was was used used toto plotplot the the analysis analysis results of SPSS software. results of SPSS software.

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Figure 6. The GA3 content of all the four (a–d) genotypes induced by COS at the concentration of 0.05 mg L−1, and ABA −1 atFigure the concentration 6. The GA3 ofcontent 2 mg of L− all1, the recorded four (a after–d) genotypes 0, 15, 30, induced and 60 days, by COS respectively. at the concentration CK represents of 0.05 control, mg L , whileand ABA the at the concentration of 2 mg L−1, recorded after 0, 15, 30, and 60 days, respectively. CK represents control, while the signifi- significance level was set as alpha = 0.05 indicated as * and alpha = 0.01 denoted as **. GraphPad Prism was used to plot the cance level was set as alpha = 0.05 indicated as * and alpha = 0.01 denoted as **. GraphPad Prism was used to plot the analysis results of SPSS software. analysis results of SPSS software. For the genotype 155202, when it was cultured on a medium supplemented with For the genotype 155202, when it was cultured on a medium supplemented with 0.05 0.05 mg L−1 COS, the IAA content was significantly increased at 30 and 60 days (Figure4b ). −1 Atmg 30 days,L COS, spherical the IAA and content torpedo was embryos significantly appeared, increased and the at number30 and 60 of days globular (Figure embryos 4b). At increased30 days, afterspherical 30 days. and Intorpedo contrast, embryos a large appeared, number of and embryoid the number bodies of were globular developed, embryos whichincreased grow after very fast.30 days. Most In cotyledon contrast, embryosa large number were shifted of embryoid to the light bodies culture were at developed, 60 days (Figurewhich3a–d). grow The very contents fast. Most of Z.T. cotyledon and GA3 embryos initially were decreased, shifted then to the enhanced light culture significantly at 60 days at(Figure 30 and 603a–d). days The for COScontents treatment of Z.T. (Figures and GA5b3 andinitially6b). decreased, then enhanced signifi- cantlyFor at the 30 genotype and 60 days of 166302, for COS the treatment content of (Figures endogenous 5b and IAA 6b). and Z.T. increased before the globularFor the embryo genotype stage of for 166302, both ABAthe content and COS, of endogenous at 30 and 60 daysIAA and (Figure Z.T.3 a–d,increasedFigure before4c , andthe Figure globular5c). Inembryo the genotype stage for 166302, both ABA the contents and COS, of GA3at 30 in and the 60 ABA days treatment (Figures initially 3a–d, 4c, increasedand 5c). andIn the then genotype decreased 166302, for the the COS contents treatment of GA3 after in 15 the days. ABA After treatment 30 days, initially an unex- in- pectedcreased decrease and then was decreased observed forfor GA3the COS and ABA;treatm however,ent after the15 days. control After and 30 COS days, treatments an unex- increasedpected decrease in almost was the sameobserved manner. for GA3 After and 60 days, ABA; the however, content ofthe GA3 control for ABA and increasedCOS treat- andments decreased increased for bothin almost control the and same COS manner. treatments After (Figure 60 days,6c). the content of GA3 for ABA increasedThe genotype and decreased BxB was for slightly both differentcontrol and when COS it comes treatments to the (Figure contents 6c). of IAA. Initially, the IAAThe content genotype was BxB high, was but slightly after 15 different days, it when dropped it comes significantly to the contents lower; of however, IAA. Ini- a significanttially, the IAA increase content in thewas IAA high, content but after was 15 observed days, it dropped at 30 and significantly 60 days for bothlower; ABA how- andever, COS a significant (Figure4d). increase The contents in the ofIAA Z.T. cont forent COS was and observed ABA increased at 30 and significantly 60 days for atboth 30ABA and 60and days COS in (Figure the given 4d). periodThe contents (Figure of5 d).Z.T. The for GA3COS contentand ABA in increased the ABA significantly and COS at 30 and 60 days in the given period (Figure 5d). The GA3 content in the ABA and COS

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treatments was lower than the control group after 30 days and was higher after 60 days (Figures 3a–d and 6d). treatments was lower than the control group after 30 days and was higher after 60 days (Figures3a–d and6d). 4.2. Germination Ability 4.2.Among Germination all the Ability four genotypes, the somatic embryo development rate of genotype 166302 Amongwas higher. all the The four cycle genotypes, of genotype the 1663 somatic02 was embryo earlier, development and the rate rateof S.E. of genotypeamong other166302 genotypes was higher. in the Thetreatment cycle ofgroup genotype was consis 166302tent was with earlier, that of and the the control rate ofgroup. S.E. amongStill, theother occurrence genotypes of S.E. in theof genotypes treatment group166302 was and consistent 155202 was with very that much of the earlier control than group. that in Still, thethe control occurrence group (Figure of S.E. of7). genotypes Genotype 166302154102 was and 1–2 155202 weeks was later very than much that earlier in the control than that group.in the Combined control group with the (Figure growth7). Genotypeof callus, it 154102 is not wasdifficult 1–2 to weeks find laterthat COS than could that in im- the provecontrol the group.callus state Combined of L. hybrid, with the when growth used of at callus, the co itncentration is not difﬁcult of 0.05 to ﬁndmg L that−1, and COS inducecould S.E. improve and ensure the callus the normal state of occurrenceL. hybrid, when of somatic used at embryos the concentration (Figure 7). of 0.05 mg L−1, and induce S.E. and ensure the normal occurrence of somatic embryos (Figure7).

Figure 7. The somatic embryo germination rate of each genotype under COS action at different Figureconcentrations, 7. The somatic with embryo control germination as C.K., and rate W denotes of each week(s).genotype under COS action at different concentrations, with control as C.K., and W denotes week(s). The callus of L. hybrid was cultured for a long time on a basic medium without adding anyThe hormones, callus of whichL. hybrid caused was slowcultured growth, for a increasedlong time wateron a basic content, medium browning, without and add- other ingphenomena, any hormones, and causedwhich caused inevitable slow stress growth, on the increased cells. Thus, water certain content, stresses browning, are formed and on otherthe cells,phenomena, and some and studies caused have inevitable shown thatstre thesess on stressesthe cells. can Thus, stimulate certain the stresses production are of formedsomatic on embryosthe cells, in and plants. some Therefore, studies have it is speculatedshown that that these the stresses early appearance can stimulate of somatic the productionembryos of genotypessomatic embryos 166302 in and plants. 155202 Theref has anore, important it is speculated relationship that the with early callus appear- status ancedeterioration. of somatic embryos To sum up,of genotypes the optimal 166302 concentration and 155202 of has COS an for important S.E. and relationship development withof L.callus hybrid statusis 0.01~0.5 deterioration. mg L− To1. Basedsum up, on the the optimal S.E. ability, concentration S.E. rate, of and COS somatic for S.E. embryo and developmentdevelopment of L. of hybrid each genotype, is 0.01~0.5 itmg was L−1. found Based thaton the the S.E. regeneration ability, S.E. abilityrate, and of somatic genotype embryo166302 development is far better thanof each all ofgenotype, the other it genotypeswas found (Figure that the8). regeneration ability of genotype 166302 is far better than all of the other genotypes (Figure 8).

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FigureFigure 8. 8. PlantletsPlantlets from from somatic somatic embryo embryo induced induced by COS, by COS, (a,b) ( asomatic,b) somatic embryos’ embryos’ germination germination plantlets of the control group under 15 and 30 days of illumination, bar = 1 mm, (c,d) somatic em- plantlets of the control group under 15 and 30 days of illumination, bar = 1 mm, (c,d) somatic bryos’ regeneration plantlets of COS under 15 and 30 days of lighting. embryos’ regeneration plantlets of COS under 15 and 30 days of lighting.

4.3.4.3. Acquisition Acquisition and and Growth Growth Potential Potential Measurement Measurement of of Somatic Somatic Embryo Embryo Regenerated Regenerated Plants Plants TheThe mature mature somatic somatic embryo embryo of of the L. hybrid and and its its base base callus callus was was transferred transferred into into a aculture culture flask flask and and gently gently flattened flattened with with forceps forceps to fullyto fully contact contact the the culture culture medium. medium. We tookWe tookfive piecesfive pieces per bottle per bottle with awith diameter a diameter of about of about 0.8 cm. 0.8 A cm. basic A mediumbasic medium was used was to used culture to culturethe germinated the germinated plantlets plantlet unders 24under◦C light 24 °C culture. light culture. After five After days, five the days, cotyledon the cotyledon embryo embryobecame became green and green developed and developed into seedling into seedling after 15–20 after days, 15–20 and days, some and immature some immature embryos embryoscontinued continued to grow andto grow developed and developed into somatic into embryo somatic germinated embryo germinated plants (Figure plants8). (Fig- ure 8).The somatic embryos induced by COS can develop into somatic embryo germinated plantsThe better somatic than embryos that of the induced control by group COS can (Figure develop8). COS into induces somatic the embryo early stagegerminated of the plantsembryo better plant than regeneration that of the and control enhances group the (Figure growth 8). development, COS induces growththe early speed, stage asof wellthe embryoas development plant regeneration situation and of plant enhances germination, the growth which development, was far better growth than speed, the as control well asgroup. development We further situation compared of plant plantlet germination, germination, which the growthwas far rate better of 2-month-old than the control plant group.growth, We and further growth compared potential, plantlet and found germinat thation, the numberthe growth of germinated rate of 2-month-old plants, growth plant growth,rate, and and development growth potential, is better and than found the control that the group. number of germinated plants, growth rate, andThe development somatic embryo is better seedling than rate the induced control bygroup. COS was significantly higher than that of theThe control somatic group embryo (80.12%), seedling and rate significant induced differencesby COS was were significantly found between higher than the treat-that − ofment the groupcontrol with group COS (80.12%), 0.01~0.1 and mg significant L 1 (Figure differences9a). In terms were of plant found growth between morphology, the treat- menta significant group with difference COS 0.01~0.1 was found mg inL−1 the (Figure number 9a). of In leaf terms blades of plant in all growth three concentrations morphology, a( Figuresignificant9b ), difference and highly was significant found in differences the number were of leaf found blades for in both all three the number concentrations of roots −1 (Figure(Figure 99b),c ) andand stemhighly length significant at the differences concentration were of found 0.05 mgfor Lboth(Figure the number9d). Further-of roots −1 (Figuremore, highly 9c) and significant stem length differences at the concentration were recorded of for0.05 plant mg L height−1 (Figure at 0.05 9d). and Furthermore, 0.1 mg L −1 highly(Figure significant9e ) and root differences length and were branching recorded rate for at plant the concentrations height at 0.05 of and 0.01 0.1 and mg0.05 L−1 mg(Figure L , 9e)respectively and root (Figurelength 9andf,g). branching It can be seen rate that at the COS concentrations can not only inhibit of 0.01 the and elongation 0.05 mg growthL−1, re- spectivelyof the hypocotyl (Figure of 9f,g). pea It induced can be seen by auxins, that COS such can as not 2, 4-d, only and inhibit IAA, the but elongation also regulates growth the

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of the hypocotyl of pea induced by auxins, such as 2, 4-d, and IAA, but also regulates the branch development and root growth of L. hybrid, which could be related to the antago- nisticbranch effect development on growth and factors root growth to some of extent,L. hybrid or, which by affecting could be auxin related synthesis to the antagonistic and polar transport.effect on growth factors to some extent, or by affecting auxin synthesis and polar transport.

FigureFigure 9. 9. GrowthGrowth vigor vigor of of L.L. hybrid hybrid inducedinduced by COS, the seedling rate rate ( (a),), number number of of leaf leaf blades blades ( (bb),), number number of of roots roots ( (cc),), stemstem length length ( (dd),), plant plant height height ( (ee),), root root length length ( (ff),), and and branching branching rate rate ( (g),), respectively. respectively. The control group waswas 3/43/4 MS MS without without addingadding COS. COS. The The significance signiﬁcance level was se sett as alpha = 0.05 0.05 indicated indicated as as * and alpha = 0.01 0.01 denoted denoted as as **. **. GraphPad GraphPad Prism Prism waswas used to plot the analysis results of SPSS software.

MeasurementsMeasurements of of the the average average number number of of leaves leaves per per branch, branch, LN/B LN/B (Figure (Figure 10d), 10d), aver- av- ageerage stem stem length length per perbranch, branch, SL/B SL/B (Figure (Figure 10e), 10ande), average and average stem or stem branch or branch diameter, diameter, BD/B (FigureBD/B (Figure 10f), of 10 L.f), hybrid of L. hybridsomaticsomatic embryo embryo regeneration regeneration plants plants showed showed that LN/B that LN/B and SL/B and ofSL/B plants of plants were werehigher higher than thanthose those of four of four branched branched plants, plants, and and BD/B BD/B was was significantly signiﬁcantly higherhigher than than that that of of plants plants with with four four branches branches (Figure (Figure 10f).10f). The The total total leaf leaf number number (L.N.) (L.N.) differencedifference between between the the four four branching branching modes modes was was highly highly significant signiﬁcant (Figure 10a).10a). Those Those withwith four four branches branches had had the the most most leaves, leaves, bu butt the the average average number number of of leaves leaves per per branch (LN/B)(LN/B) was was the the least least (Figure (Figure 1010a,d).a,d). It isis speculatedspeculated thatthat the the increase increase in in the the total total number number of ofleaves leaves of of the the plant plant may may increase increase the the competition competition between between branches branches for for the the light light environ- envi- ronment,ment, which which reduces reduces the the photosynthetic photosynthetic capacity capacity of theof the leaves leaves and and eventually eventually limits limits the theaccumulation accumulation of material. of material. The SL/BThe SL/B of the ofL. the hybrid L. hybriddecreased decreased with the with number the ofnumber branches, of branches,which may which result may from result the plant’sfrom the nutrition plant’s distribution.nutrition distribution.

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FigureFigure 10. Effects ofof COSCOS on on branching branching development development of L.of hybrid,L. hybrid, the totalthe total number number of leaves of leaves (a), stem (a), length stem (lengthb), the ( averageb), the averagenumber number of leaves of per leaves branch per (c branch), average (c), no average of average no of leaves average per leaves branch per (d), branch average (d stem), average length stem per branchlength (pere), andbranch average (e), andstem average diameter stem per diam brancheter ( fper). The branch signiﬁcance (f). The levelsignificance was set level as alpha was =set 0.05 as indicatedalpha = 0.05 as *indicated and alpha as = * 0.01and denotedalpha = as0.01 **. denotedGraphPad as Prism**. GraphPad was used Prism to plot was the used analysis to plot results the analysis of SPSS results software. of SPSS software.

5.5. Discussion Discussion ItIt is is well-known well-known that that oligosaccharides oligosaccharides can can stimulate stimulate or or inhibit inhibit growth growth and and develop- develop- mentment in in different different plants plants [56]. [56]. In In the the wheat wheat cr crop,op, the the number number of of spikes, spikes, grains grains per per spike, spike, andand total total grain grain yield yield were were increased increased by by spraying spraying COS COS at at the the tillering tillering stage stage [53]. [53]. Moreover, Moreover, COSCOS has low-costlow-cost production production and and has has a number a numb ofer promising of promising biological biological properties, properties, namely namelybiodegradability, biodegradability, biocompatibility, biocompatibility, and non-allergenicity and non-allergenicity [57]. In [57]. plants, In plants, the mechanism the mech- anismof chitosan of chitosan is yet tois beyet fully to be understood. fully understood. Chitosan, Chitosan, with its with oligomers, its oligomers, has been has widely been widelyused in used plants in toplants confer to resistanceconfer resistance against against abiotic stresses,abiotic stresses, such as such water as deficit, water salinity,deficit, salinity,and heat and stress heat [58 stress], and enhances[58], and photosyntheticenhances photosynthetic activities as activities well as increases as well as the increases chitinase theactivity chitinase in seedlings activity in by seedlings 30–50% [ 59by]. 30–50% These characteristics[59]. These characteristics make COS valuablemake COS for valua- many bleindustries, for many such industries, as cosmetology, such as food,cosmetology, biotechnology, food, pharmacology,biotechnology, andpharmacology, medicine [60 and,61]. medicineOur hypothesis [60,61]. that Our COS hypothesis could positively that COS impactcould positively the growth impact and development the growth and of L. devel- hybrid opmentleads to of promising L. hybrid results. leads to The promising optimal results. concentration The optimal of somatic concentration embryos induced of somatic by COSem- −1 −1 bryosin L. hybridinducedis 0.05by COS mg Lin L.. Thehybrid addition is 0.05 of mg 0.05 L−1 mg. The L additionof exogenous of 0.05 mg COS L can−1 of increaseexogenous the COSrate andcan increase number the of somatic rate and embryos, number ultimatelyof somatic promotingembryos, ultimately somatic embryos’ promoting growth somatic and L. hybrid embryos’development. growth The and number development. of somatic The embryos number of in somatic callus of embryos inwas callus higher of L. inhybrid solid medium supplemented with COS, and the average number of somatic embryos per gram was higher in solid medium supplemented with COS, and the average number of somatic of callus was up to a maximum of 2500 in two genotypes of L. hybrid. embryos per gram of callus was up to a maximum of 2500 in two genotypes of L. hybrid. The present work shows that after COS treatment, 2000~2500 individual embryos The present work shows that after COS treatment, 2000~2500 individual embryos were produced on average per gram of callus, significantly different from the basic medium were produced on average per gram of callus, significantly different from the basic me- (3/4 MS) (Figure1a–d). Statistical analysis of the number of mature somatic embryos dium (3/4 MS) (Figure 1a–d). Statistical analysis of the number of mature somatic embryos showed that the trend was consistent with the number of total embryos, and the somatic showed that the trend was consistent with the number of total embryos, and the somatic embryos developed well (Figures2a–d and3). In this study, the S.E. number was signifi- embryos developed well (Figures 2a–d and 3). In this study, the S.E. number was significantly enhanced 2–3-fold by COS induction compared with control or without any plant cantly enhanced 2–3-fold by COS induction compared with control or without any plant growth regulators. In tobacco, the oligo-chitosan can be precisely bound to the walls and growth regulators. In tobacco, the oligo-chitosan can be precisely bound to the walls and

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the membranes of tobacco cells, which increases the accumulation of IAA and decreases the concentration of IAA peroxidase in the cells in suspension to promote plant growth and development [62,63]. Alginate oligosaccharides regulate auxin-related genes’ expression to accelerate auxin biosynthesis and transport and reduce IAA oxidase activity and induce calcium signaling in the roots of rice [64]. In this study, different genotypes have different S.E. abilities and different sensitivity to COS. In general, COS induces the efficiency of somatic embryos of L. hybrid. Among all the four genotypes, 166302 produced better results in response to COS 0.05 mg L−1 when added to the culture medium. Statistics of somatic embryo regeneration plants induced by COS showed that somatic embryos developed well under COS induction. Most of them were able to grow into typical regenerated plants. The somatic embryo seedling rate was as high as 82.14%, which was significantly higher than that of the control group. The root length of somatic embryo germinated plants induced by COS was significantly longer than that of the control group. The number of leaves, plant height, stem length, and rooting number were also significantly different from those of the control group, indicating that COS is beneficial to the radical development of a somatic embryo and root growth elongation somatic embryo germinated plants. In plants, the rapid metabolism, light isomerization in vitro, complicated synthetic routes, and too expensive cost hinder ABA use [65]. Compared to ABA, COS is economical, diverse, and can be utilized in many advanced ways [66]. The enzymatic process to obtain COS was considered the perfect and environment-friendly process with low cost, which controls the DA and degree of polymerization (D.P.) of the products. COS is easily soluble in neutral water compared to ABA, which is insoluble in water [67]. Most importantly, COS significantly induces S.E., and improved tolerance of low- temperature stress, U.V., and salt stress in soybean [68], and in our study, in L. sino- americanum. In our study, the statistical data of somatic embryo regeneration plants induced by COS showed that somatic embryos developed well under COS induction. Most of them were able to grow into normal germinated plants. Signaling induced by the chitosan molecule comprises specific cellular receptors that are transduced by secondary messenger(s) such as reactive oxygen species (ROS), H2O2, nitric oxide (NO), and phyto- hormones inside the cell to induce physiological responses. Its inhibitory effects of radicals by chitosan treatment has been stated [69]. The branching rate of somatic embryo germinated plants induced by COS was significantly higher. When the COS concentration was 0.1 mg L−1, the two-month-old somatic embryo germinated plant had the highest branching rate, as high as 18.32%, which was significantly different from the 4.91% of the control group (p < 0.01). The branching rate of somatic embryo germinated plants with a COS concentration of 0.05 mg L−1 was 14.92%, which was higher than the control group. The average number of leaves per branch, stem diameter, and the average stem length of each branch decreased with an increase in the number of branches.

6. Conclusions COS increased the efﬁcacy of somatic embryogenesis when used at a particular concentration, such as 0.05 mg L−1. Our results showed that adding a certain concentration of COS to the growth medium could promote the growth of stem and root of regenerated plantlets in somatic embryos of L. hybrid. The plant height was 19.21% higher than that of the control group, and the stem increased by 17.36%, the stem diameter increased by 18.03%, and the root increased by 36.12%. Highly signiﬁcant differences were observed for leaf growth and root thickening when the concentration was 0.05 mg L−1.

Author Contributions: Data curation, A.A. and M.Z.; L.S.; statistical analysis, Formal analysis, S.u.R.; Funding acquisition, J.S. and J.C.; Investigation, S.H. and J.Z.; Writing—original draft, A.A and T.C.; Writing—review and editing, J.C. All authors have read and agreed to the published version of the manuscript. Forests 2021, 12, 557 15 of 17

Funding: This work was supported by the Nature Science Foundation of China (32071784), the Dis- tinguished Professor Project of Jiangsu province, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data for the manuscript can be provided at the request of readers. Acknowledgments: The authors are grateful for the support from the Nature Science Foundation of China (32071784), Distinguished Professor Project of Jiangsu province, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Conﬂicts of Interest: The authors declare that they have no conﬂict of interest.

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